Detection, identification, quantification, and characterization of biomolecules or cells of interest, such as stem cells or cancer cells, through testing of biological samples is an important aspect in the fields of medical diagnostics and medical research. Biological solutions, such as blood, spinal fluid, cell culture and urine, are routinely analyzed for their microscopic particle concentrations.
As an example, for determining cell concentrations in biological solutions, a commonly used method is to spread a cell-containing solution into a thin layer without cell overlap in the vertical direction. A precise volume is determined by keeping the height of the solution at a known constant level. Cells are viewed under an optical microscope and enumerated in defined areas. To eliminate the variation caused by microscopes, an area-defining grid is preferred in the counting chamber. A commonly used cell counting device is called hemacytometer, as disclosed in Risch (U.S. Pat. No. 1,693,961) and Hausser et al. (U.S. Pat. No. 2,039,219).
More recently, cell counting has been accomplished using flow cytometry, a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. A beam of light, e.g., laser light, of a single wavelength is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at a point where the stream passes through the light beam; one in line with the light beam and several perpendicular to it. Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a higher wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector it is then possible to derive various types of information about the physical and chemical structure of each individual particle. Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of fluorescence, scattered light, and transmitted light for each cell.
In addition to prohibitive cost ($150,000 to $500,000) for a flow cytometry system, there are many technical problems associated with flow cytometry. For example, many technical problems result from cells clumping and clogging or sticking in the nozzle of the flow cytometer, causing the stream of fluid to deflect and become misaligned with the optics. Also, resulting aerosolization of the sample prevents biohazardous samples, e.g. human blood cells potentially infected with HIV or hepatitis virus, from being sorted unless stringent precautions are taken.
Further, flow cytometry can only provide an indirect measure of cell concentration and cell size. Flow cytometry relies on mixing a sample with a known concentration of beads, determining the number of beads that pass the detector in the flow cytometer, and correlating the bead count with the number of cells that pass the detector to determine the concentration of cells in the sample.
There is an unmet need for efficient and cost-effective systems and methods for counting biomolecules and cells.